scholarly journals Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

1998 ◽  
Vol 9 (12) ◽  
pp. 3561-3578 ◽  
Author(s):  
Harri Palokangas ◽  
Ming Ying ◽  
Kalervo Väänänen ◽  
Jaakko Saraste
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Marker Proteins ◽  
Golgi Region ◽  
Intermediate Compartment ◽  
Acidic Compartments

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ∼80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

Effects of bafilomycin A1 on cytosolic pH of sheep alveolar and peritoneal macrophages: evaluation of the pH-regulatory role of plasma membrane V-ATPases.

1995 ◽  
Vol 198 (8) ◽  
pp. 1711-1715 ◽  
Author(s):  
T A Heming ◽  
D L Traber ◽  
F Hinder ◽  
A Bidani
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Initial Rate ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Phi Regulation

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

scholarly journals The discrepancy between presenilin subcellular localization and γ-secretase processing of amyloid precursor protein

2001 ◽  
Vol 154 (4) ◽  
pp. 731-740 ◽  
Author(s):  
Philippe Cupers ◽  
Mustapha Bentahir ◽  
Katleen Craessaerts ◽  
Isabelle Orlans ◽  
Hugo Vanderstichele ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Precursor Protein ◽  
Subcellular Compartment ◽  
Marker Proteins ◽  
Intermediate Compartment ◽  
The Relationship

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

scholarly journals RUFY3 and RUFY4 are ARL8 effectors that couple lysosomes to dynein-dynactin

2021 ◽  
Author(s):  
Tal Keren-Kaplan ◽  
Amra Saric ◽  
Saikat Ghosh ◽  
Chad Williamson ◽  
Rui Jia ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Serum Starvation ◽  
Anterograde Transport ◽  
Fyve Domain ◽  
Axon Development ◽  
Necessary And Sufficient

Abstract The small GTPase ARL8 associates with lysosomes and recruits several effectors that mediate coupling to kinesins for anterograde transport, as well as tethering for eventual fusion with other organelles. Herein we report the identification of the “RUN- and FYVE-domain-containing” proteins RUFY3 and RUFY4 as novel ARL8 effectors that couple lysosomes to dynein-dynactin for retrograde transport. Using various biochemical approaches, we find that RUFY3/4 interact with both GTP-bound ARL8 and dynein-dynactin. In addition, we show that RUFY3/4 are both necessary and sufficient for concentration of lysosomes in the juxtanuclear area of the cell. RUFY3/4 also promote retrograde transport of lysosomes in the axon of hippocampal neurons. The function of RUFY3/4 in retrograde transport is required for juxtanuclear redistribution of lysosomes upon serum starvation or cytoplasmic alkalinization, and may underlie the reported roles of RUFY3/4 in axon development/degeneration, cancer and immunity. These studies thus establish RUFY3/4 as novel ARL8-dependent, dynein-dynactin adaptors, and highlight the role of ARL8 in the regulation of both anterograde and retrograde lysosome transport.  

scholarly journals The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Diego Huet ◽  
Thierry Blisnick ◽  
Sylvie Perrot ◽  
Philippe Bastin
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Anterograde Transport ◽  
Cargo Transport ◽  
Cilia And Flagella ◽  
Bidirectional Movement

The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.

scholarly journals ER exit sites in Drosophila display abundant ER-Golgi vesicles and pearled tubes but no megacarriers

2021 ◽  
Author(s):  
Ke Yang ◽  
Min Liu ◽  
Zhi Feng ◽  
Marta Rojas ◽  
Lingjian Zhou ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Fat Body ◽  
Retrograde Transport ◽  
Imaginal Discs ◽  
Membrane Structures ◽  
Molecular Events ◽  
Golgi Region ◽  
Er Exit Sites ◽  
Essential Questions ◽  
Intermediate Compartment

AbstractSecretory cargos are collected at ER exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, we used transgenic tagging in Drosophila flies, 3D-SIM and FIB-SEM to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs and imaginal discs overexpressing ERES determinant Tango1. We found in front of ERES a pre-cis-Golgi region involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate intermediate compartment or collection of large carriers, for which we found no evidence. We found, however, many vesicles, as well as pearled tubules connecting ERES and Golgi.

scholarly journals Rab7a and Mitophagosome Formation

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 224 ◽  
Author(s):  
Esther Tan ◽  
Bor Tang
Keyword(s):  
Retrograde Transport ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Autophagosome Formation ◽  
Guanine Nucleotide Exchange ◽  
Gtpase Activating Proteins ◽  
Retromer Complex ◽  
Nucleotide Exchange Factor

The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.

scholarly journals Biochemical and structural insights into Rab12 interactions with RILP and its family members

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jana Omar ◽  
Efrat Rosenbaum ◽  
Adi Efergan ◽  
Bayan Abu Sneineh ◽  
Adva Yeheskel ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Mast Cell ◽  
Single Molecule ◽  
Secretory Granules ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
The Other ◽  
Dynamics Simulations ◽  
Structural Insights

AbstractAlongside its biosynthetic functions, the small GTPase Rab12 negatively regulates mast cell (MC) exocytosis by its interaction with RILP to promote retrograde transport of the MC secretory granules. Given the role of Rab effectors in mediating Rab functions, in this study we used biochemical and in silico tools to decipher Rab12 interactions with its RILP family effectors. We show that Rab12 interacts with RILP, RILP-L1 and RILP-L2 independently of each other, whereby lysine-71, in mouse Rab12, is critical for Rab12 interactions with RILP-L1 or RILP-L2, but is dispensable for the binding of RILP. Focusing on RILP, and relying on molecular dynamics simulations, functional mutational analyses and peptide inhibition assays, we propose a model for the Rab12-RILP complex, consisting of a RILP homodimer and a single molecule of active Rab12, that interacts with the RILP homology domain (RHD) of one RILP monomer and a C-terminal threonine in the other monomer via its switch I and switch II regions. Mutational analyses of RILP RHD also demonstrate its involvement in the regulation of MC secretory granule transport. Jointly, our results provide structural and functional insights into the Rab12-RILP complex on the basis of which new tools could be generated for decoding Rab12 functions.

scholarly journals Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

2021 ◽  
Author(s):  
Rui Yan ◽  
Kun Chen ◽  
Ke Xu
Keyword(s):  
Protein Trafficking ◽  
Volume Ratio ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Fine Tuning ◽  
En Bloc ◽  
Golgi Transport ◽  
Receptor Interactions ◽  
Cargo Receptor ◽  
Intermediate Compartment

The endoplasmic reticulum (ER)-to-Golgi transport is critical to protein secretion and intracellular sorting. Cargo carriers mediating the ER-to-Golgi transport are morphologically diverse, but it remains unclear whether this diversity arises from different cargo receptors, or whether it could lead to differential transport kinetics. Here we report a tubular ER-Golgi intermediate compartment (t-ERGIC) that is induced by the cargo receptor SURF4 and selectively expedites the ER-to-Golgi transport of SURF4 cargoes. Lacking the canonical ERGIC marker ERGIC-53 yet positive for the small GTPase Rab1, the t-ERGIC is further distinct from the stereotypical vesiculo-tubular cluster (VTC) ERGIC by its extremely elongated shape (~10 um long with <30 nm diameter). With its exceptional surface-to-volume ratio and en bloc cargo packaging, high (~2 um/s) intracellular traveling speeds, and ER-Golgi recycling capability, the t-ERGIC provides an efficient means for trafficking SURF4-bound cargoes. The biogenesis and cargo selectivity of t-ERGIC both depend on SURF4, which recognizes the N-terminus of soluble cargoes and co-clusters with the selected cargoes to expand the ER exit site. At the steady state, the t-ERGIC-mediated fast ER-to-Golgi transport is antagonized by retrograde transport based on the cargo C-terminal ER retrieval signal: we thus demonstrate the fine-tuning of protein trafficking and localization via its primary structure. Together, our results argue that specific cargo-receptor interactions give rise to distinct transport carriers, which in turn regulate the ER-to-Golgi trafficking kinetics.

scholarly journals Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

1997 ◽  
Vol 137 (2) ◽  
pp. 319-333 ◽  
Author(s):  
Jochen Scheel ◽  
Rainer Pepperkok ◽  
Martin Lowe ◽  
Gareth Griffiths ◽  
Thomas E. Kreis
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Marker Proteins ◽  
Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive trans-Golgi-associated compartment

2000 ◽  
Vol 278 (2) ◽  
pp. F317-F326 ◽  
Author(s):  
Corinne E. Gustafson ◽  
Toshiya Katsura ◽  
Mary McKee ◽  
Richard Bouley ◽  
James E. Casanova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Hormonal Stimulation ◽  
Intracellular Location ◽  
Membrane Location ◽  
Recycling Pathway ◽  
Intracellular Vesicles

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK1 cells even in the absence of hormonal stimulation. Incubating cells at 20°C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H+-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20°C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.

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